A long-pursued goal of structural biology has been the determination of the structure of membrane proteins in their native membrane environment, and in their various functional states as defined by transmembrane gradients of substrate molecules and membrane potentials. Application of such gradients, let alone crystallization in a membrane environment, has not been possible in the case of X-ray structure determination. The application of gradients has remained out of reach as well for the analysis of 2D crystals by electron crystallography. The proposed work concerns the further development of a general method for single-particle cryo-EM structure determination of membrane proteins reconstituted into liposomes. The method is called random spherically- constrained (RSC) single-particle reconstruction. In the proposed work we will work to surmount several remaining technical hurdles, and then apply this combination of biochemistry, EM imaging and image processing methods toward a nanometer- resolution structure of the GluA2 glutamate receptor as reconstituted into a lipid membrane.